1. Field of the Invention
The present invention relates generally to the field of gene therapy for cancer. More specifically, the present invention presents a method of controlling the expression of therapeutically valuable gene products via inducible promoters. The present invention provides a method whereby induced gene expression in the intended cell targets is enhanced and prolonged in a spatially and temporally regulable manner by means of heat or light inducible promoters. Moreover, the present invention provides a method whereby the background gene expression in non-targeted cells is reduced or eliminated.
2. Description of the Related Art
One of the major obstacles to the success of chemotherapy and radiation therapy for cancer is the difficulty in achieving tumor-specific cell killing. The inability of radiation or cytotoxic chemotherapeutic agents to distinguish between tumor cells and normal cells necessarily limits the dosage that can be applied. As a result, diseases relapse due to residual surviving tumor cells is frequently observed.
The use of gene therapy in cancer treatment presents many of the same disadvantages as chemotherapy and radiation therapy. Problems with current state-of-the-art gene therapy strategies include the inability to deliver the therapeutic gene specifically to the target cells. This leads to toxicity in cells that are not the intended targets. For example, manipulation of the p53 gene suppresses the growth of both tumor cells and normal cells, and intravenous administration of tumor necrosis factor alpha (TNFxcex1) induces systemic toxicity with such clinical manifestations as fever and hypertension.
Attempts have been made to overcome these problems. These include such strategies as: the use of tissue-specific receptors to direct the genes to the desired tissues (Kasahara, N., et al., Science, 266:1373-1376 (1994)), the use of tissue-specific promoters to limit gene expression to specific tissues (e.g. use of the prostate specific antigen promoter) and the use of heat (Voellmy R., et al., Proc. Natl. Acad. Sci. USA, 82:4949-4953 (1985)) or ionizing radiation inducible enhancers and promoters (Trainman, R. H., et al., Cell 46: 567-574 (1986); Prowess, R., et al., Proc. Natl. Acad. Sci. USA 85, 7206-7210 (1988)) to enhance expression of the therapeutic gene in a temporally and spatially controlled manner. The heat inducible heat shock protein (HSP) promoter has been used to direct the expression of genes such as the cytokine IL-2.
Weichselbaum and colleagues were the first to discover the radiation inducible response of the early growth response (Egr-1) gene promoter. Accordingly, they have attempted to direct expression of such cytotoxic genes as TNF-xcex1 to tumor cells to enhance radiation cell killing by means of this promoter. Previously, systemic administration of the cytokine TNF-xcex1 as an adjuvant to ionizing radiation was initially reported to result in enhanced killing in a mouse xenograft tumor system. It has since been shown partially effective in human tumors. The effect of TNFxcex1 appears to be dosage-dependent, as its tumor-killing effect correlates with its serum concentration. However, systemic toxicity of TNFxcex1 restricts the dosage that can be applied and thus limits the usefulness of the treatment regimen. Attempts have also been made to deliver the TNFxcex1 gene to tumor cells via adenoviral vector and/or liposomes. Unfortunately, expression of the TNFxcex1 gene is not restricted to the tumor sites due to the xe2x80x98leakinessxe2x80x99 of the promoter.
In an attempt to localize the level of TNFxcex1 to the general area of radiation exposure and thereby reduce systemic toxicity, Weichselbaum and colleagues employed the radiation inducible Egr-1 promoter to activate the TNFxcex1 gene in situ. Earlier studies showed that the expression of certain immediate-early genes such as jun/fos and Egr-1 are activated in cells exposed to ionizing radiation (Sherman, M. L., et al., Proc. Natl. Acad. Sci. USA, 87: 5663-5666 (1997); Hallahan, D. E., et al., Proc. Natl. Acad. Sci. USA, 88: 2156-2160 (1991)). By placing the TNFxcex1 gene under the control of the Egr1 promoter (EGRp), the expression of the TNFxcex1 is enhanced in those cells harboring an EGRp-TNFxcex1 plasmid when exposed to ionizing radiation. In vivo, the serum level of TNFxcex1 is greatly enhanced (Weichselbaum R. R., et al., Cancer Res. 54: 4266-4269 (1994)) within a few hours after irradiation. The combined treatment with this plasmid and radiation leads to a partial regression of a xenografted tumor during the course of the treatment. The level of TNFxcex1 dropped precipitously within 24 hours; further decreases in serum level of TNFxcex1 coincided with regrowth of the tumors.
There are several possible reasons for the recurrence of the tumor upon cessation of therapy. The most obvious reason is probably the same limitation seen with chemotherapy or radiation therapy in general, viz., insufficient dosage levels. A major problem, which limits the amount of TNFxcex1 produced, is the weak and transient nature of the Egr-1 promoter. This promoter is intrinsically weak, with a maximum of less than three-fold increase in expression upon induction. Moreover, the induced expression is of necessity transient. This, coupled with the weakness of the promoter, permits only a brief exposure of the tumor cells to the TNFxcex1.
Another factor that limits the production of sufficient dosage of TNFxcex1 is that not every tumor cell will have taken up the TNFxcex1 plasmid. While it has been suggested that repeated administration may help to improve the treatment outcome, it is not clear if the repeated delivery of a suboptimal low dosage of TNFxcex1 will be useful, the problems posed by an immune response notwithstanding. Although it might be conceivable to deliver larger doses of plasmids, the problem of promoter leakiness has hindered such an approach. It is known that a substantial basal level of activity (20-30%) can be detected with the Egr-1 promoter even in the absence of ionizing radiation (Weichselbaum, et al., supra). This is not surprising, as the radiation response element, a CArG box, is part of the serum response element.
The HSP promoter is also rather leaky. In the absence of heat, this promoter exhibits a 25-30% background level of expression, not suitable for most cytotoxic genes. As this level of expression will be harmful to unirradiated normal cells that take up the gene. Hence, administration of this plasmid has been restricted to small doses of intra-tumoral injections to minimize systemic toxicity.
Therefore, while it may be advantageous to employ a spatially and temporally regulated promoter such as the HSP and Egr-1 promoters to enhance specificity of gene expression at the site of heat or radiation treatment, current versions of those promoters have serious problems that restrict their applicability. In order to apply these promoters for use in cancer therapy, it is necessary to eliminate or greatly reduce background expression in unheated or unirradiated cells. Ideally, the expression of cytotoxic genes should be limited to the area of external stimuli (heat or radiation). Additionally, to ensure a sufficient level of expression of therapeutic genes, the weak and transient nature of gene expression driven by these promoters must be improved.
It is important to note that even when an improved inducible vector system which can restrict the expression of a therapeutic gene to the area of external stimuli is developed, there is still the problem of expression in normal heated or irradiated bystander cells. Thus, it is critical to be able to further restrict the expression of therapeutic genes only to the intended targets, e.g., tumor cells.
The prior art is deficient in the lack of effective means of inhibiting unwanted toxic side effects of gene therapy treatments for cancer, as well as providing a method for enhancing and sustaining gene expression in targeted tumor cells in a controllable manner. The present invention fulfills this longstanding need and desire in the art.
The current invention provides the composition and methods for the controlled activation of DNA molecules for gene therapy. Activation of these DNA molecules leads to the production of protein products which then may provide opportunities for therapeutic manipulation of cells containing said DNA molecules. This may be achieved via alterations in cell growth and metabolism of the targeted cells and may include effects on neighboring cells via secretion of therapeutic products. The invention offers the options of sustained activation or activation regulable by the application of antibiotics. The invention further provides novel expression vectors for use in gene therapy of local and metastatic breast, ovarian and prostate cancer.
An original strategy to confine and enhance therapeutic gene expression to tumors spatially and temporally is also presented, in the form of an expression vector designed for use in local and metastatic breast, ovarian and prostate cancer.
In one embodiment of the present invention, there is provided a method for sustained and enhanced expression of a gene via activation of a heat or light inducible promoter. In a modification of this method, heat or light is used to activate the promoter, but continued levels of gene expression are modulated by concentrations of an antibiotic (tetracycline or its derivatives), acting on a fusion protein with a tetracycline-responsive element.
In yet another embodiment of the present invention, there is provided a method of constructing the vectors for gene therapy activation modalities.
In another embodiment of the present invention, there are provided improved vectors for reducing background expression in unheated and unirradiated cells.
In another embodiment of the present invention, there are provided improved vectors for reducing expression in heated and irradiated normal bystander cells.
In another embodiment of the present invention, there are provided expression vectors for use in gene therapy treatment of local and metastatic breast and ovarian cancer.
In another embodiment of the present invention, there are provided expression vectors for use in gene therapy treatment of local and metastatic prostate cancer.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.